The present invention relates to a method for synthesis of acrylamide derivatives, preferably immobilines. The method is especially suitable for synthesis of the immobiline acrylamido agmatine but may be used for synthesis of any acrylamide derivative. The compounds synthesised by the method can be used in the production of a separation material. A preferred example is a separation material for separating amphoteric compounds based on differences in isoelectric points (pI), in particular by electrophoresis (isoelectric focusing, IEF). Another preferred example is a separation material for 2D electrophoresis.
In two-dimensional, 2D, electrophoresis substances are separated in two dimensions. In the first dimension, the substances are separated by isolectric focusing. Isoelectric focusing is often followed by a second dimension of gel electrophoresis in which each separated substance is further separated according to molecular weight and/or molecular size. Isoelectric focusing and gel electrophoresis is typically run in two dimensions which are perpendicular to each other. 2D electrophoresis can be run both in analytical and preparative amounts of substances to be separated.
The substances to be separated are typically bio-organic and encompass primarily compounds having polypeptide structure and/or carbohydrate structure. Proteins are particularly important.
In certain kinds of electrophoresis the carrier material has been functionalized with groups which provide conditions that are beneficial for the intended separation. One important kind of groups has been pH-buffering groups. By immobilising pH-buffering groups of different pKa's between the anode end and the cathode end of an electrophoretic gel it became possible during the late seventies to set up immobilised pH-gradients (Aminkemi, U.S. Pat. No. 4,130,470) to be used in isoelectric focusing. In order to have good pH-gradients it was important to have a range of different pH-buffering groups with increasing/decreasing pKa values spaced within a desired pH-interval. The difference between the pKa of two neighbouring buffering groups has typically been 1-2 pH units. For pH intervals extending above pH 10 there has been described a charged carrier material in WO 02/25264 (Amersham Biosciences) having improved stability against hydrolysis at pH>10 for the manufacture of separation materials. This document describes
(1) properly selecting immobilised pH-buffering groups amongst those that have
                (a) a pH-dependent charge on a nitrogen atom which binds to an sp2-hybridised carbon atom, and        (b) a pKa≧9.5, e.g. ≧10.0 or ≧10.5 or ≧11.0, and/or(2) utilising carrier materials, which have been based on certain acryl monomers.        
Immobilines are acrylamide derivatives with buffering groups. Immobilines are weak acids or bases defined by their pK value. A preferred use of the immobilines is for 2D electrophoresis. One of the most interesting immobilines is acrylamido agmatine. The synthesis of acrylamido agmatine as described in WO 02/25264 involves the reaction of agmatine sulphate and barium hydroxide in water. Thereafter, the formed precipitate of barium sulphate has to be removed by filtration and desalted agmatine is obtained by freeze drying of the aqueous phase. To get rid of most of the barium sulphate, a precipitation has to be repeated at least once. Next step is redissolving desalted agmatine in an organic solvent and addition of acryloyl chloride and a base. The reaction is time consuming and often gives poor yield and purity. Furthermore, the work-up of the reaction mixture often faces difficulties to remove the base and the excess of unreacted acryloyl chloride (often hydrolysed to acrylic acid during the course of reaction).
Acrylamido agmatine has a theoretical pKa value of 13.52 and suitable for preparation of separation materials for separation in pH intervals extending 10.